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High yield yellow fever virus strain with increased propagation in cells

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Title: High yield yellow fever virus strain with increased propagation in cells.
Abstract: The invention provides a an inactive, non-replicating vaccine comprising whole virion, chemically inactivated Yellow Fever virus which is inactivated using a method that ensures preservation of critical, neutralizing epitopes. The Yellow Fever virus has been adapted to propagate in cells to higher yields than the unadapted virus. The invention also provides methods for preventing Yellow Fever viral infection. ...


Browse recent Xcellerex, Inc. patents - Marlborough, MA, US
USPTO Applicaton #: #20110287519 - Class: 4352351 (USPTO) - 11/24/11 - Class 435 
Chemistry: Molecular Biology And Microbiology > Virus Or Bacteriophage, Except For Viral Vector Or Bacteriophage Vector; Composition Thereof; Preparation Or Purification Thereof; Production Of Viral Subunits; Media For Propagating



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The Patent Description & Claims data below is from USPTO Patent Application 20110287519, High yield yellow fever virus strain with increased propagation in cells.

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RELATED APPLICATION

This application is a continuation-in-part of copending International Application No. PCT/US2010/043010, filed on Jul. 23, 2010, which claims the benefit of priority to U.S. Provisional Application No. 61/230,483, filed on Jul. 31, 2009. The entire teachings of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The Yellow Fever virus is endemic, that is, continuously present with low levels of infection in some tropical areas of Africa and the Americas, where it regularly amplifies into epidemics. Other parts of the world, including coastal regions of South America, the Caribbean islands, and Central and North America, are infested with the mosquito vector capable of transmitting the virus and are therefore considered at risk for yellow fever epidemics (World Health Organization Fact Sheet No. 100, revised December, 2001).

For example, in Africa alone, thirty-three countries with a combined population of 508 million, are at risk (Id.). Each year, the World Health Organization (WHO) estimates there are 200,000 cases of yellow fever, with 30,000 deaths (Id.). Travel to these tropical regions also is believed to result in a small number of imported cases in countries generally free of yellow fever. Although yellow fever cases have not been reported in Asia, “this region is at risk because the appropriate primates and mosquitoes are present” (Id.).

The Yellow Fever (YF) virus is in the genus Flavivirus, in the family Flaviviridae. In the so-called “jungle” or “sylvan cycle”, the YF virus is enzootic, maintained and transmitted by canopy breeding mosquitoes to monkeys in the rainforests. The “urban cycle” begins when humans become infected by entering the rainforests and are bitten by YF-infected mosquitoes. The “urban cycle” continues with peridomestic transmission from humans to mosquitoes and thence to other humans, and can result in yellow fever epidemics in villages and cities. Illness ranges in severity from a self-limited febrile illness to severe hepatitis and fatal hemorrhagic disease.

Unvaccinated humans, including both native people and travelers to YF endemic areas are at significant risk of YF infection when occupational and other activities bring them in contact with infected mosquitoes in the sylvan cycle or the urban cycle.

Patients with yellow fever may be viremic, i.e., have virus in their blood, for 3 to 6 days during the early phase of illness. This phase may be followed by a short period of symptom remission.

The toxic phase develops as the fever returns, with clinical symptoms including, for example, high fever and nausea, hemorrhagic symptoms, including hematemesis (black vomit), epistaxis (nose bleed), gum bleeding, and petechial and purpuric hemorrhages (bruising). Deepening jaundice and proteinuria frequently occur in severe cases.

In the late stages of disease, patients can develop hypotension, shock, metabolic acidosis, acute tubular necrosis, myocardial dysfunction, and cardiac arrhythmia. Confusion, seizures, and coma can also occur, as well as complications such as secondary bacterial infections and kidney failure.

There is no specific treatment for yellow fever. Steps to prevent yellow fever include use of insect repellent, protective clothing, and vaccination with the available, but risky attenuated vaccine.

Live, attenuated vaccines produced from the 17D substrain, are available, but adverse events associated with the attenuated vaccine can lead to a severe infection with the live 17D virus, and serious and fatal adverse neurotropic and viscerotropic events, the latter resembling the severe infection by the wild-type YF virus. Thus there is a need for a safer, inactivated, non-replicating vaccine that will elicit a neutralizing antibody response while eliminating the potential for neurotropic and viscerotropic adverse events.

Thus, there is an on-going need for an effective, inactivated, “killed” or non-replicating vaccine in order to avoid the potential for neurotropic and viscerotropic adverse events associated with the currently available attenuated YF 17D vaccine. Further, there is a need for an improved vaccine produced in Vero cells without animal-derived proteins, a vaccine that can be safely used for persons for whom the live vaccine is contraindicated or for whom warnings appear on the label. Such individuals include immuno-suppressed persons, persons with thymic disease, egg-allergic, young infants, and the elderly.

A problem with any potential inactivated virus is that it may need to be delivered at a higher titer than the existing live attenuated vaccines, because the latter can expand antigenic mass during cycles of replication in the host whereas an inactivated vaccine contains a fixed dose of antigen. Therefore, in order to develop a sufficiently potent inactivated vaccine, it is desirable to modify the YF virus in order to produce a high yield of virus in the conditioned medium (also called supernatant fluid) of a cell culture. It is highly desirable to use the attenuated 17D vaccine strain for vaccine manufacturing, since the 17D strain can be manipulated at a lower level of biocontainment than the wild-type virulent YF virus. However, the attenuated 17D vaccine strain yields in cell culture are inherently lower than yields of wild-type virus. For these reasons, modifications of the 17D vaccine strain to achieve higher yields in cell culture used for vaccine production would be useful.

BRIEF

SUMMARY

OF THE INVENTION

The invention provides a vaccine comprising a strain or strains of Yellow Fever virus which have been adapted to propagate in Vero cells to higher yields than an unadapted virus. “Unadapted virus” is defined to mean that Yellow Fever virus vaccine known as 17D. Sequence analysis of examples of such strains demonstrates that an adapted virus possessing a mutation in the envelope (E) protein resulting in a lysine to arginine substitution in amino acid residue 160 has improved properties. The invention also provides for vaccines comprising a Yellow Fever virus containing one or more mutations in the E protein, that result in increased propagation in Vero cells and in higher yields when using serum free culture medium than the unadapted virus.

Additional examples of adapted Yellow Fever virus strains which propagate in Vero cells to higher yields than unadapted virus have been identified. These include modified Yellow Fever virus strains wherein the nucleic acid molecules of said modified Yellow Fever virus strains comprise at least one amino acid mutation selected from: an amino acid mutation in the NS1 protein, an amino acid mutation in the NS2A protein, and an amino acid mutation in the NS4B protein, optionally wherein said at least one amino acid mutation is in further combination with an amino acid mutation in the envelope protein. Preferred embodiments include 1) a strain having three mutations: a) a lysine to arginine substitution in amino acid residue 160 (lys160arg) in the E protein, b) a threonine to isoleucine substitution in amino acid residue 317 (thr317ile) in the non-structural protein 1 (NS1), and c) a phenylalanine to leucine substitution in amino acid residue 170 (phe170leu) in the non-structural protein 2A (NS2A); and 2) a strain with a mutation in the non-structural protein 4B (NS4B), resulting in an isoleucine to methionine substitution at amino acid residue 113 (ile113met).

The invention provides for vaccines comprising a Yellow Fever virus containing one or more mutations selected from: a mutation in the NS1 protein optionally combined with a mutation in the E protein; a mutation in the NS2A protein optionally combined with a mutation in the E protein; and a mutation in the NS4B protein optionally combined with a mutation in the E protein that result in increased propagation in Vero cells and in higher yields than the unadapted virus.

The Yellow Fever virus is the prototype species in the genus Flavivirus, in the family Flaviviridae. Structural and functional studies of the E protein of tick-borne encephalitis (TBE) virus, a fast-growing, virulent member of the flavivirus genus, indicate that Domains I and II in the E protein of TBE participate in an acidic pH-dependent conformational change that facilitates flavivirus membrane fusion with the host and subsequent infectivity. The junction of Domains I and II function as a ‘molecular hinge’ resulting in a major rearrangement of these domains from of the normal dimeric structure of the E protein at acid pH into a homotrimeric state. [Rey F A et al. The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution. Nature 375: 291-298 (1995); Heinz F X et al. Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology 198: 109-117 (1994); Mandl C W et al. Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model. Journal of Virology 63(2): 564-571 (1989); Harrison S C. Viral membrane fusion. Nature structural and molecular biology 15(7): 690-698 (2008); Stiasny K et al. Molecular mechanisms of flavivirus membrane fusion. Amino acids DOI 10.1007/s00726-009-0370-4, published on line 1 Nov. 2009.]

Lys 160 in the E protein of Yellow Fever virus is located in the molecular hinge region between Domains I and II. Mutations in this region could alter the acid-dependent conformational change in region Domain I of the E protein required for fusion and virus internalization into the cell cytoplasm. Without being bound by theory, higher yields seen with the lysine to arginine change at amino acid 160 in Domain I of the E protein of the adapted Yellow Fever virus strain may be due to an increased affinity for protons that arginine provides as compared with lysine, that results in enhanced membrane fusion with the host and more efficient infectivity. In regard to the invention, it is important to note that the side chains of lysine and arginine have pKa values of 10.53 and 12.48, respectively, indicating a one hundred fold greater affinity for protons in arginine than in lysine. The increased affinity for protons that the side chain of arginine shows relative to lysine's side chain may enhance the rate and efficiency of E protein conformational change at the molecular hinge, membrane fusion, and flavivirus infectivity, resulting in higher yields of virus in the adapted virus strain.

Other members within the genus Flavivirus include West Nile, dengue, and Japanese encephalitis viruses. The non-structural proteins found in West Nile Virus are known to be directly or indirectly involved in viral RNA synthesis. Amino acid substitutions in the non-structural proteins of these viruses have been shown to affect the yields of mutant viruses grown in Vero cells. For example, a proline to leucine substitution at amino acid 250 in the NS1 protein of the flavivirus Kunjin, a West Nile Virus subtype, grows at 100-fold lower titers than wild-type virus. Similarly, mutation of the C-terminal sites in the NS2A protein of yellow fever virus was shown to be lethal for virus replication. Brinton Mass. The molecular biology of west nile virus: a new invader of the western hemisphere. Annual Review of Microbiology 56: 371-402 (2002).

In a first aspect, the invention provides a modified Yellow Fever virus strain that results in increased propagation in Vero cells and a higher yield in the conditioned medium of a cell culture relative to the unadapted virus comprising at least one mutation relative to the unadapted virus selected from: a mutation in the E protein, a mutation in the NS1 protein, a mutation in the NS2A protein, and a mutation in the NS4B protein, optionally wherein said at least one mutation in the NS1 protein, the NS2A protein, or the NS4B protein is in further combination with a mutation of the E protein.

Replacement of basic amino acids that are located within 20 amino acids, or within 10 Angstroms, of lysine 160 in the E protein of the Yellow Fever virus (including lysine 160 itself), with amino acids having higher side chain pKa values than the replaced basic amino acids, can result in strains of Yellow Fever virus that produce higher yields of virus than an unadapted Yellow Fever virus. The invention thus provides for Yellow Fever viruses, and vaccines containing them, comprising a modified nucleic acid molecule encoding an E protein, the virus being capable of propagating in Vero cells to higher yields than the unadapted virus. Preferred embodiments include viruses comprising a modified E protein with an increased pKa within 20 amino acids, or within 10 Angstroms, of lysine 160 in the E protein.

In a third aspect, the invention provides a nucleic acid molecule comprising a sequence encoding a modified envelope protein of the Yellow Fever virus, wherein said nucleic acid molecule comprises a nucleotide mutation in the codon for the amino acid at position 160 of the envelope protein. In an embodiment of this aspect, the invention provides a nucleic acid molecule comprising a sequence encoding at least one modified nucleic acid relative to the nucleic acid of the unadapted virus, wherein said at least one modified nucleic acid is selected from: a modified nucleic acid of the NS1 protein, a modified nucleic acid of the NS2A protein and a modified nucleic acid of the NSB4 protein, optionally wherein said at least one modified nucleic acid is in further combination with a modified nucleic acid of the envelope protein of the Yellow Fever virus, wherein said optional modified nucleic acid of the envelope protein comprises a nucleotide mutation in the codon for the amino acid at position 160 of the envelope protein. In a further embodiment of this aspect, the nucleotide mutation in the codon for the amino acid at position 160 of the envelope protein results in a change from AAG to AGG, AGA, CGC, CGA, CGG or CGU. Additionally, the invention provides for vectors, constructs, modified Yellow Fever virus strains, and cells comprising or containing such a nucleic acid molecule or a protein encoded thereby.

In a fourth aspect, the invention provides a modified Yellow Fever virus strain, wherein the nucleic acid molecule of said strain comprises a sequence encoding an envelope protein of the Yellow Fever virus, wherein said envelope protein comprises an amino acid mutation at position 160 of the envelope protein. In an embodiment of this aspect, the invention provides a modified Yellow Fever virus strain, wherein the nucleic acid molecule of said strain comprises a sequence encoding an envelope protein of the Yellow Fever virus, wherein said envelope protein optionally comprises an amino acid mutation at position 160 of the envelope protein.

In a fifth aspect, the invention provides a nucleic acid molecule comprising a sequence encoding an envelope protein of the Yellow Fever virus, wherein said envelope protein comprises an amino acid mutation at position 160 of the envelope protein. In an embodiment of this aspect, the invention optionally provides a nucleic acid molecule comprising a sequence encoding an envelope protein of the Yellow Fever virus, wherein said envelope protein comprises an amino acid mutation at position 160 of the envelope protein. Additionally, the invention provides for vectors, constructs, modified Yellow Fever virus strains, and cells comprising or containing such nucleic acid molecules or proteins encoded thereby. The nucleic acid molecules preferably comprise a sequence encoding a modified envelope protein of the Yellow Fever virus, wherein said nucleic acid molecule encodes the protein sequence in SEQ ID NO. 4, 6, or 7.

In a sixth aspect, the invention provides a method for enhancing the propagation of Yellow Fever virus in cells. In an embodiment of this aspect, the method comprises mutating a nucleic acid molecule comprising a sequence encoding the envelope protein of the Yellow Fever virus, wherein the mutation comprises a nucleotide mutation in the codon for the amino acid at position 160 of the envelope protein. In another embodiment of this aspect, the method optionally comprises mutating a nucleic acid molecule comprising a sequence encoding the envelope protein of the Yellow Fever virus, wherein the mutation comprises a nucleotide mutation in the codon for the amino acid at position 160 of the envelope protein. In a further embodiment, the method comprises mutating a nucleic acid molecule comprising a sequence encoding the envelope protein of the Yellow Fever virus, wherein said mutation comprises an amino acid mutation at position 160 of the envelope protein. In a final embodiment, the method optionally comprises mutating a nucleic acid molecule comprising a sequence encoding the envelope protein of the Yellow Fever virus, wherein said mutation comprises an amino acid mutation at position 160 of the envelope protein. The word “mutating” is intended to mean selecting for a mutation, or introducing a mutation. The relevant mutant viruses can be obtained by a method of selection and evolutionary pressure during passages in a specific host cell line (such as Vero cells) or by site-directed mutagenesis using infectious clone technology well known in the art. However, the former method is preferred because it identifies mutated viruses by virtue of the desired phenotypic characteristic (increased yields in Vero cell cultures).

In a seventh aspect, the invention provides a modified Yellow Fever virus strain, wherein the nucleic acid molecule of said strain comprises a nucleotide mutation in the codon for amino acids flanking the E160 codon selected from position 134, 137, 144, 148, 157, 160, 175, or 177 of the envelope protein of Yellow Fever virus. In an embodiment of this aspect, the invention provides a modified Yellow Fever virus strain, wherein the nucleic acid molecule of said strain optionally comprises a nucleotide mutation in the codon for amino acids flanking the E160 codon selected from position 134, 137, 144, 148, 157, 160, 175, or 177 of the envelope protein of Yellow Fever virus. In another embodiment of this aspect, the mutated codon within 20 amino acids flanking the E160 mutation results in an amino acid mutation in the envelope protein at that position, wherein the pKa value of the side chain of the mutated amino acid is higher than the pKa value of the side chain of the original amino acid at that position.

In an eighth aspect, the invention provides for Yellow Fever viruses, and vaccines containing them, comprising a modified nucleic acid molecule encoding an NS1 protein, the virus being capable of propagating in Vero cells to higher yields than the unadapted virus. Preferred embodiments include viruses comprising a modified NS1 protein and a modified E protein. A more preferred embodiment includes viruses comprising a modified NS1 protein and a modified E protein with an increased pKa within 20 amino acids, or within 10 Angstroms, of lysine 160 in the E protein.

In a ninth aspect, the invention provides for Yellow Fever viruses, and vaccines containing them, comprising a modified nucleic acid molecule encoding an NS2A protein, the virus being capable of propagating in Vero cells to higher yields than the unadapted virus. Preferred embodiments include viruses comprising a modified NS2A protein and a modified E protein. A more preferred embodiment includes viruses comprising a modified NS2A protein and a modified E protein with an increased pKa within 20 amino acids, or within 10 Angstroms, of lysine 160 in the E protein.

In a tenth aspect, the invention provides for Yellow Fever viruses, and vaccines containing them, comprising a modified nucleic acid molecule encoding an NS1 protein and an NS2A protein, the virus being capable of propagating in Vero cells to higher yields than the unadapted virus. Preferred embodiments include viruses comprising a modified NS1 protein, a modified NS2 protein, and a modified E protein. A more preferred embodiment includes viruses comprising a modified NS1 protein, a modified NS2 protein, and a modified E protein with an increased pKa within 20 amino acids, or within 10 Angstroms, of lysine 160 in the E protein.

In an eleventh aspect, the invention provides for Yellow Fever viruses, and vaccines containing them, comprising a modified nucleic acid molecule encoding an NS4B protein, the virus being capable of propagating in Vero cells to higher yields than the unadapted virus.

In a twelfth aspect, the invention provides a nucleic acid molecule comprising a sequence encoding a modified non-structural protein 1 of the Yellow Fever virus, wherein said nucleic acid molecule comprises a nucleotide mutation in the codon for the amino acid at position 317 of the non-structural protein 1. In an embodiment of this aspect, the nucleotide mutation in the codon for the amino acid at position 317 of the non-structural protein 1 results in a change from ACA to AUA. Additionally, the invention provides for vectors, constructs, modified Yellow Fever virus strains, and cells comprising or containing such a nucleic acid molecule or a protein encoded thereby.

In a thirteenth aspect, the invention provides a nucleic acid molecule comprising a sequence encoding a modified non-structural protein 2A of the Yellow Fever virus, wherein said nucleic acid molecule comprises a nucleotide mutation in the codon for the amino acid at position 170 of the non-structural protein 2A. In an embodiment of this aspect, the nucleotide mutation in the codon for the amino acid at position 170 of the non-structural protein 2A results in a change from UUU to CUU. Additionally, the invention provides for vectors, constructs, modified Yellow Fever virus strains, and cells comprising or containing such a nucleic acid molecule or a protein encoded thereby.

In a fourteenth aspect, the invention provides a nucleic acid molecule comprising a sequence encoding a modified non-structural protein 4B of the Yellow Fever virus, wherein said nucleic acid molecule comprises a nucleotide mutation in the codon for the amino acid at position 113 of the non-structural protein 4B. In an embodiment of this aspect, the nucleotide mutation in the codon for the amino acid at position 113 of the non-structural protein 4B results in a change from AUA to AUG. Additionally, the invention provides for vectors, constructs, modified Yellow Fever virus strains, and cells comprising or containing such a nucleic acid molecule or a protein encoded thereby.

In a fifteenth aspect, the invention provides a modified Yellow Fever virus strain, wherein the nucleic acid molecule of said strain comprises a sequence encoding proteins of the Yellow Fever virus, wherein said proteins comprise an amino acid mutation at position 160 of the envelope protein, at position 317 of the NS1 protein, at position 170 of the NS2A protein, or at position 113 of the NS4B protein.

In a sixteenth aspect, the invention provides a nucleic acid molecule comprising a sequence encoding an envelope protein, an NS1 non-structural protein, an NS2A non-structural protein, or an NS4B non-structural protein of the Yellow Fever virus, wherein said proteins comprise an amino acid mutation at position 160 of the envelope protein, at position 317 of the NS1 protein, at position 170 of the NS2A protein, or at position 113 of the NS4B protein. Additionally, the invention provides for vectors, constructs, modified Yellow Fever virus strains, and cells comprising or containing such a nucleic acid molecule or proteins encoded thereby. The nucleic acid molecules preferably comprise a sequence encoding a modified protein of the Yellow Fever virus, wherein said nucleic acid molecule encodes the protein sequence in SEQ ID NO: 7 and SEQ ID NO: 8.

In a seventeenth aspect, the invention provides a method for enhancing the propagation of Yellow Fever virus in cells. In an embodiment of this aspect, the method comprises mutating a nucleic acid molecule comprising a sequence encoding the envelope protein, the NS1 non-structural protein, the NS2A non-structural protein, or the NS4B non-structural protein of the Yellow Fever virus, wherein said mutations comprise an amino acid mutation at position 160 of the envelope protein, at position 317 of the NS1 protein, at position 170 of the NS2A protein, or at position 113 of the NS4B protein. The word “mutating” is intended to mean selecting for a mutation, or introducing a mutation.

In an eighteenth aspect, the invention provides a modified Yellow Fever virus strain, wherein the nucleic acid molecule of said strain comprises a nucleotide mutation in the codon for amino acids 317 of the NS1 protein, 170 of the NS2A protein, or 113 of the NS4B protein, and wherein the nucleic acid molecule also comprises a nucleotide mutation in the codon for amino acids flanking the E160 codon selected from position 134, 137, 144, 148, 157, 160, 175, or 177 of the envelope protein of Yellow Fever virus. In an embodiment of this aspect, the mutated codon within 20 amino acids flanking the E160 mutation results in an amino acid mutation in the envelope protein at that position, wherein the pKa value of the side chain of the mutated amino acid is higher than the pKa value of the side chain of the original amino acid at that position.

The invention also provides methods of making and using the nucleic acid molecules, modified E proteins, modified NS1 proteins, modified NS2A proteins, modified NS4B proteins, modified Yellow Fever viruses, vectors, constructs and cells containing the same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the passage history of Vero cells during the manufacture of the disclosed yellow fever vaccine.

FIG. 2 is a schematic representation of the preparation of the virus seeds.

FIG. 3A is a schematic of the process used for 10 serial passages (P1 through P10) to modify the nucleotide sequence of the viral genome virus to develop a seed virus with enhanced growth in Vero cells for preparation of an inactivated Yellow Fever virus candidate.

FIG. 3B is a graphical representation of the virus replication for passage one (P1) and passage 11 (P11) of the initial experiment, in which P11 virus differs from P1 by a single mutation at E160 (lys→arg)

FIG. 3C is a graphical representation of a repeat passaging study of passage one (Bp1, Cp1) and passage 11 (B-p11, C-p11) virus performed in a series of experiments: Series B and C.

FIG. 4A-L depicts the consensus alignment of the P1 and P11 nucleic acid sequences. The starting nucleic acid sequence, P1, is identified herein as SEQ ID NO: 1. A comparison of the P1 passage and the P11 passage revealed a genetic mutation at nucleotide residue #211 of SEQ ID NO: 1, and a second mutation at nucleotide residue #1452 of SEQ ID NO: 1. Thus, “P1 consensus” corresponds to SEQ ID NO.1; “P11 consensus” corresponds to SEQ ID NO. 2 having the codon mutation at envelope protein amino acid position 160.

FIG. 5A-J depicts the amino acid sequence of P1 and P11, with the Series B-P1 and Series B3-P11 amino acid sequences from the repeat passaging study. The amino acid sequence for P1 is identified herein as SEQ ID NO: 3. A comparison of the amino acid sequence for P1 and that of P11 (SEQ ID NO. 4) revealed a mutation at amino acid residue 160 of the envelope protein (E160) (amino acid 445 of the P1 amino acid sequence in FIG. 5B). Series B-P1 and Series B3-P11 present partial amino acid sequences from the repeat passaging study. The amino acid sequence for B-P1 is identified herein as SEQ ID NO: 5. A comparison of the amino acid sequence for B-P1 and that of B3-P11 (SEQ ID NO. 6) revealed a mutation at amino acid residue 160 of the envelope protein (E160) in B3-P11 (amino acid 445 of the P1 amino acid sequence).

FIG. 6 depicts the comparative 50% plaque reduction neutralization test (PRNT50) titers between treatment groups of BALB/c and CD-1 strain mice in a preliminary mouse study (M-9003-002) of the efficacy of inactivated Yellow Fever vaccine.

FIG. 7 is a graphical representation of PRNT50 antibody titers for the preliminary mouse study (M-9003-002).

FIG. 8A-EE depicts the consensus alignment of the P1, B-P1, C-P1, B3-P11 and C1-P11 nucleic acid sequences. The nucleic acid sequence, P1, is identified herein as SEQ ID NO: 15. The nucleic acid sequence, B-P1, is identified herein as SEQ ID NO: 9. The nucleic acid sequence, B3-P11, is identified herein as SEQ ID NO: 11. The nucleic acid sequence, C-P1, is identified herein as SEQ ID NO: 10. The nucleic acid sequence, C1-P11, is identified herein as SEQ ID NO: 12. A comparison of the B-P1 passage and the B3-P11 passage revealed a genetic mutation in B3-P11 at nucleotide residue #1452 of SEQ ID NO: 15, a second mutation in B3-P11 at nucleotide residue #3402 of SEQ ID NO: 15, and a third mutation in B3-P11 at nucleotide residue #4016 of SEQ ID NO: 15. A comparison of the C-P1 passage and the C1-P11 passage revealed a genetic mutation in C1-P11 at nucleotide residue #7225 of SEQ ID NO: 15. SEQ ID NO: 11 corresponds to B3-P11, and has the codon mutations at envelope protein amino acid position 160, non-structural protein 1 amino acid position 317, and non-structural protein 2A amino acid position 170. SEQ ID NO.12 corresponds to C1-P11, and has the codon mutation at non-structural protein 4B amino acid position 113.

FIG. 9A-F depicts the amino acid sequence of B-P1 and B3-P11 from the repeat passaging study. The amino acid sequence for B-P1 is identified herein as SEQ ID NO: 13. A comparison of the amino acid sequence for B-P1 and that of B3-P11 (SEQ ID NO: 7) revealed a mutation at amino acid residue 160 of the envelope protein (E160) (amino acid 445 in FIG. 9A), a mutation at amino acid residue 317 of the non-structural protein 1 (NS1-317) (amino acid 1095 in FIG. 9B), and a mutation at amino acid residue 170 of the non-structural protein 2A (NS2A-170) (amino acid 1300 in FIG. 9C). Series B-P1 and Series B3-P11 present complete amino acid sequences from the repeat passaging study.

FIG. 10A-F depicts the amino acid sequence of C-P1 and C1-P11 from the repeat passaging study. The amino acid sequence for C-P1 is identified herein as SEQ ID NO: 14. A comparison of the amino acid sequence for C-P1 and that of C1-P11 (SEQ ID NO: 8) revealed a mutation at amino acid residue 113 of the non-structural protein 4B (NS4B-113) (amino acid 2369 in FIG. 10E). Series C-P1 and Series C1-P11 present complete amino acid sequences from the repeat passaging study.

DETAILED DESCRIPTION

OF THE PREFERRED EMBODIMENTS

A description of preferred embodiments of the invention follows. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. At the outset, the invention is described in its broadest overall aspects, with a more detailed description following. The features and other details of the compositions and methods of the invention will be further pointed out in the claims.

Overview of Approach and Benefits

The invention relates to compositions and methods for use in preventing Yellow Fever virus infection. Disclosed herein is a method of producing an inactivated Yellow Fever virus candidate, the method comprising the serial passage of the YF 17D virus (i.e., an “unadapted virus”) in certified African green monkey kidney cells (VERO) to increase the titer to yield a sufficient antigenic mass to induce a protective immune response and/or modify the nucleotide sequence of the viral genome. This method has been repeated and shown to be reproducible.

One embodiment of the invention is a modified Yellow Fever (YF) virus that will grow to high titers in Vero cells. Another embodiment of the invention is a vaccine comprising a whole virion, chemically inactivated Yellow Fever (YF) virus prepared from serum-free conditioned medium from Vero cells infected with 17D virus. In one embodiment of the invention, the virus has been purified from host cell DNA and proteins by depth filtration, ultrafiltration, diafiltration, and chromatographic separation. The method is described in U.S. Application Ser. No. 61/228,026 filed on Jul. 23, 2009, and its corresponding International Application No. PCT/US2010/043013 filed on Jul. 23, 2010, which are each incorporated herein by reference. The purified virus may be inactivated by using a method that ensures preservation of critical, neutralizing epitopes. For example, the virus can be inactivated using formalin, heat, UV, gamma irradiation or beta-propiolactone. A purified, inactivated virus may be formulated with an adjuvant, such as adsorbed to aluminum hydroxide adjuvant, and stored as a liquid at temperatures of from about 2 degrees Celsius (2° C.) to about 8 degrees Celsius (8° C.).

A vaccine containing the purified, inactivated virus is believed to be safer than the currently available attenuated, live YF virus vaccine because the disclosed inactivated YF virus vaccine is non-replicating. The inventors of the present subject matter have now developed a safer, inactivated, non-replicating YF vaccine that will elicit a neutralizing antibody response while eliminating the potential for neurotropic and viscerotropic adverse events. In addition, the improved vaccine can be manufactured by modern methods in Vero cells without animal derived proteins, and therefore it can be used safely in persons (including egg-allergic persons) for whom the live vaccine (produced in hens\' eggs) is contraindicated or for whom warnings appear in the label. Such warnings would include, for example warnings to immunosuppressed persons, persons with thymic disease, egg-allergic persons, infants <9 months, and the elderly.

Adaptation of Yellow Fever Virus for Robust Production in Vero Cells:

The Vero cells used in the virus development phase were obtained from the World Health Organization (W.H.O.) seed lot, WHO Vero 10-87 Cell Bank at Passage 134. The WHO Vero 10-87 Cell Bank was originally made by the Institut Merieux using the ATCC Vero cell line CCL81 at Passage 129. The cells were thawed into OptiPRO™ SFM (serum-free medium) supplemented with 5% fetal bovine serum which was removed 24 hours later and replaced with OptiPRO™ SFM medium without fetal bovine serum. The serum, certified as being of USA origin, was gamma irradiated and had been tested for adventitious agents by the manufacturer; additional testing for sterility, mycoplasma, and adventitious viruses was performed on this material by WuXi AppTec. All subsequent passages of Vero cells to make the cell banks, virus seeds, and vaccine were made in OptiPRO™ SFM without serum. No other animal derived materials or products were used in producing the cell banks or the final vaccine according to an embodiment of the invention.

Preparation of Vero Cell Banks:

Master and Working Cell banks were prepared according to cGMP and were tested and characterized according to FDA Points to Consider. The Vero cells had an established provenance and were free from regulatory concerns about Bovine spongiform encephalitis (BSE). Serum-free growth medium was employed in propagating cells.

Passage History of Vero Cells During Manufacture of Seed Viruses and Vaccine Lots:

The passage history of Vero cells during the manufacture of the disclosed yellow fever vaccine is shown schematically in FIG. 1. The WHO cells were received at Passage 134, the Master Cell Bank (MCB) and Working Cell bank (MWCB) were banked at Passages 139 and 143 respectively. The cells were further expanded a maximum of 11 passages to Passage 154 during cell expansion in stationary cultures prior to seeding of the bioreactor used for virus production. The estimated number of population doublings in the bioreactor is calculated to be 1 to 3.

Preparation of Master and Working Virus Seeds:

FIG. 2 is a schematic representation of the preparation of Virus seeds according to an embodiment of the invention. An important safety factor for the disclosed vaccine is the use of the attenuated YF 17D vaccine for manufacture. The attenuated virus used as a starting material was a commercial vaccine, YF-VAX® (Sanofi Pasteur, Swiftwater Pa.) which had undergone various tests for adventitious agents. The original YF-VAX® material used to inoculate Vero cells was derived from embryonated hens\' eggs, and contained hydrolyzed porcine gelatin as a stabilizer. However, the likelihood of carry-over of an adventitious agent from eggs was mitigated by use of RNA transfection to produce the Pre-Master Virus Seed.

The cells were propagated in OptiPro-SFM medium (Invitrogen, Grand Island, N.Y.). To develop the modified Yellow Fever (YF) virus that will grow to high titers in Vero cells, initially the YF-17D virus at a 0.01 multiplicity of infection (MOI) was used to infect a T-25 flask with a confluent layer of Vero cells. The cell culture was incubated at 37° C. and 5 percent CO2.

Once cytopathic effect (CPE) was observed in about 2+(50%) of the cells, aliquots of the culture were prepared, labeled as passage one (P1) and stored at −80° C. for use as the inoculum to continue the serial passages. A schematic of the procedure used to make P1 through P10 is shown in FIG. 3A.

An aliquot of the Passage 1 (P1) virus was diluted 10−1 through 10−8 and each dilution was inoculated onto confluent monolayers of three (3) Vero cell cultures propagated in sterile 12 well plates from which growth medium had been removed.

Log10 dilutions were prepared by transferring 0.2 ml of virus to 1.8 ml of phosphate buffered saline (PBS) to equal a 10−1 dilution. The virus plus PBS was mixed and then a new pipette was used to transfer 0.2 ml to 1.8 ml of PBS=10−2, and then repeated through 10−8 dilution. Twelve well confluent monolayers of Vero cell culture were labeled and log10 dilutions of the P1 material (negative control, 10−1 (3 wells), 10−2 (3 wells), 10−3 (3 wells), 10−4 (3 wells), 10−5 (3 wells), 10−6 (3 wells), 10−7 (3 wells) and 10−8 (3 wells) were prepared and inoculated onto medium-free cultures using a new pipette for each dilution of inoculum. The negative control cultures were inoculated with a similar volume of PBS. After inoculating the cultures they were incubated at 37° C. for 1 hour with intermittent rocking and then 1.0 ml of maintenance medium was added per culture. Cells were observed each day for cytopathic effect (CPE) and recorded as 1+(25% of the cell monolayer effected), 2+(50% of the cell monolayer effected), 3+(75% of the cell monolayer effected) and 100% (all of the cell monolayer effected). Estimates of CPE were based on a comparison with the control cells. The plaque assay was also performed on the same dilutions of inoculum to verify that the CPE represented viral infectivity.

Once CPE (2+) developed in these cultures, five 0.5 ml aliquots of the medium were harvested from the cultures that received the highest dilution or next to the highest dilution of inoculum. The five aliquots were prepared and stored as passage 2 (P2) at −80° C. The strategy was to select the virus population that replicated at or near the highest log10 dilution based on the appearance of CPE in the cells. As such, the virus population selected would be the population that was best adapted to replicate in the cells with possible genetic changes that will allow for an increase in viral titer.

Subsequently, log10 dilutions were prepare of an aliquot of the P2 virus and used to infect cultures of Vero cell propagated in 12-well plates as described for passage one YF virus. Similar methods were employed to complete 10 serial passages of the virus.

P10 and P11:

At each serial passage, each of the aliquots used as the inoculum was also tested to determine the infectivity titers by plaque assay in Vero cells. At passage 10, five single, well isolated plaques, each representing progeny from a single infectious virus particle, were selected at the highest dilution that yielded plaques. Each plaque was suspended in 0.3 ml of medium containing Human Serum Albumin (HSA) to protect the virus infectivity during freezing and stored at −80° C.

The series of passages (P1 to P10) of the YF 17D virus in Vero static cultures at dilutions of 10−1 to 10−8 were performed at the University of Texas Medical Branch (Galveston, Tex.). The strategy was to select the virus population that replicated at or near the highest log10 dilution based on the microscopic appearance of CPE in the Vero cells. The virus population that showed cytopathic effects at the highest dilution, the P10 harvest, was selected as the optimized, “high-yield” virus. The high yield virus population that showed CPE at the highest dilution was sequenced.

The High Yield Virus:

The “high yield” virus was adapted for increased replication in Vero cells by 10 serial virus passages at terminal dilution in Vero cells. At Virus Passage 10, a single plaque forming unit was picked and passed in fluid culture to produce a mini-seed stock at Virus Passage 11. The graph in FIG. 3B shows comparative growth curves of P1 and P11 viruses, that had been inoculated at high MOI; the data indicate that the P11 virus has a higher peak titer than the P1 virus. This virus (P11) showed a 3-7 fold increased replication capacity in in Vero cells compared to the YF 17D at Virus Passage 1. The Virus Passage 11 virus stock was used for RNA extraction and the RNA used to produce cGMP grade virus seeds.

RNA Sequence of the Vero Adapted 17D Virus (P11)

The full genomic consensus sequences of the viruses at P1 and P11 from the original YF-VAX® were determined. Two genetic mutations or nucleotide differences were found, as shown in Table 1 below. One nucleotide difference lies in the capsid (C) gene and one in the envelope (E) gene. The term “capsid” as used herein, refers to the shell of protein that surrounds and protects the nucleic acid of a virus. The change in the C gene was silent (no amino acid change), whereas the E gene mutation resulted in an amino acid (Lys→Arg) mutation.

TABLE 1

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stats Patent Info
Application #
US 20110287519 A1
Publish Date
11/24/2011
Document #
13012917
File Date
01/25/2011
USPTO Class
4352351
Other USPTO Classes
International Class
12N7/01
Drawings
74


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Chemistry: Molecular Biology And Microbiology   Virus Or Bacteriophage, Except For Viral Vector Or Bacteriophage Vector; Composition Thereof; Preparation Or Purification Thereof; Production Of Viral Subunits; Media For Propagating